Malaria caused by Plasmodium falciparum (Pf) results in more than 250 million clinical cases, and nearly one million deaths annually. A vaccine would be the ideal intervention for reducing malaria morbidity and mortality. All clinical manifestations and pathology of malaria are caused by the erythrocytic stage of the parasite life cycle, and thus all sequelae of malaria disease begin when the parasite invades erythrocytes. Blocking parasite invasion of erythrocytes would prevent parasite replication and all clinical disease. Pf parasites invade erythrocytes by binding to specific erythrocyte receptors. Thus, blocking parasite invasion of erythrocytes by inducing antibodies that interfere with parasite receptor-ligand interaction during invasion is an important approach to malaria vaccine development. A well-studied Pf ligand is EBA-175 that binds its receptor sialic acids on glycophorin A. Antibodies to EBA-175 can block parasite invasion. Unfortunately there are strains of Pf that invade by alternate pathways not involving sialic acids. Development of vaccines that effectively block invasion must thus induce antibodies against multiple ligands, antibodies that interfere with the sialic acid and alternate pathways of invasion. The reticulocyte binding homolog protein family (PfRH) of proteins has been identified to play a major role in binding and invasion of erythrocytes by alternate pathways excluding sialic acids. We aim to assess if antibodies induced by immunization with the PfRH proteins when combined with antibodies against EBA-175 can effectively block invasion of parasites into erythrocytes. Assessments will be systematically performed using blocking of erythrocyte binding and parasite growth invasion inhibition assays. We will first express recombinant candidate proteins to raise antibodies against these candidates in rabbits. The candidates PfRH 1, 2b, 4 and 5, together with EBA-175 will be assessed. Our immediate goal is to potently interfere with parasite binding and invasion into erythrocytes using the strategy of a multi-ligand vaccine that induces antibodies that block multiple pathways of invasion. Interfering on multiple fronts with the single crucial step of erythrocyte invasion is at the core of our innovation and approach. We will select the best combination of candidates and propose to develop them in Phase II as a multi-ligand, invasion blocking vaccine. Thus in Phase II we will create producer clones of the selected recombinant candidates and systematically assess them in rhesus monkeys with multiple adjuvant formulations suitable for human use, down select the best adjuvant formulation(s), and produce material under cGMPs in preparation for clinical trials designed to determine the efficacy of this multi-ligand merozoite invasion blocking vaccine. PUBLIC HEALTH RELEVANCE: Malaria causes 400-500 million clinical cases and nearly 1 million deaths annually, and is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Protein Potential's goal is to develop and commercialize a >90% protective malaria vaccine for primary markets with a potential for >$1 billion annual revenues; 1) travelers from the developed world, and 2) infants, young children, and adolescent girls in the developing world. Success in this project will significantly decrease the cost of development and reduce time to market for an effective malaria vaccine.